Inflammatory gout: observations over a half-century.

This is a discussion of acute gouty arthritis, seen for over 50 years of engagement. It addresses the evolution of our current understanding of the interaction between urate crystals and key cellular components of the gouty inflammatory paroxysm, with new material on pathogenesis.

Tonic inhibition of chemotaxis in human plasma.

We found exaggerated chemotaxis in plasma treated with EDTA and thought that the EDTA might itself be inhibiting a tonic inhibitor(s) of chemotaxis. Our plasma fractionations suggested that evidence should be sought for a lipid moiety carrying this activity, and on spectrometry (LC-MS-MS together with GC-MS analyses), the biologically active but not the inactive fraction contained oleic and arachidonic acids. Because fatty acids are largely protein bound, we flooded plasma preparations with delipidated albumin, reasoning that it would bind enough fatty acids, including inhibitory ones, to counter their tonic inhibition. Indeed, we observed dramatic increases in chemotaxis. Hence, adding delipidated albumin to plasma has a similar effect to that of adding EDTA--amplification of the chemotactic response. Oleic acid in physiologic concentrations diminishes the magnifying effects of both EDTA and of delipidated albumin, and in fact diminishes the chemotactic response even without the presence of the amplifiers of chemotaxis. In contrast, arachidonic acid amplifies further the effect of EDTA but not of delipidated albumin, and this augmentation appears to be caused by an EDTA-dependent enrichment of the chemotactic gradient with leukotriene B4 (LTB4). We conclude that oleic acid, the blood levels of which vary among individuals, is at least one tonic inhibitor of chemotaxis in plasma.

Dysregulation of TLR3 impairs the innate immune response to West Nile virus in the elderly.

West Nile virus (WNV), a mosquito-borne flavivirus, has recently emerged in North America, and the elderly are particularly susceptible to severe neurological disease and death from infection with this virus. We have investigated the innate immune response of primary human macrophages to WNV in vitro and have found significant differences between the responsiveness of macrophages derived from younger donors and that from older donors. Binding of the glycosylated WNV envelope protein to the C-type lectin dendritic cell-specific intercellular adhesion molecule 3 (ICAM3) grabbing nonintegrin (DC-SIGN) leads to a reduction in the expression of Toll-like receptor 3 (TLR3) in macrophages from young donors via the signal transducer and activator of transcription 1 (STAT1)-mediated pathway. This signaling is impaired in the elderly, and the elevated levels of TLR3 result in an elevation of cytokine levels. This alteration of the innate immune response with aging may contribute to the permeability of the blood-brain barrier and suggests a possible mechanism for the increased severity of WNV infection in older individuals.

Phagocytic function of human blood polymorphonuclear leukocytes in the presence of carrageenan, a potential vaginal microbicide.

Carrageenan is currently undergoing clinical trials as the active constituent of a vaginal gel product for use as a female-controlled option to prevent the transmission of HIV during sexual intercourse. Here we show that in the presence of 0.5 mg/ml of carrageenan, human blood polymorphonuclear leukocytes (PMN) do not ingest this material, as evidenced by a lack of progressive vacuolization, but can ingest microorganisms present in the medium, excluding adjacent carrageenan. Moreover, PMN move at normal speeds, respond chemotactically, and reduce nitroblue tetrazolium (NBT) to formazan on stimulation. Hence, in the presence of carrageenan the phagocytic response appears to remain intact.

Chemotactic activity of human blood leukocytes in plasma treated with EDTA: chemoattraction of neutrophils about monocytes is mediated by the generation of NAP-2.

In slide preparations of human blood leukocytes in autologous plasma containing EDTA, many adherent monocytes are initially chemotactic for neutrophils (PMN). We have identified the chemotactic factor that they generate as neutrophil-activating peptide-2 (NAP-2), as evidenced by distraction of the gradient by authentic human NAP-2, the importance of platelets in the media, which elaborate the precursor of NAP-2, and suppression of the chemotactic response by serine protease inhibitors, which would block the monocyte-derived serine esterase that creates NAP-2 from its immediate precursor. Consistent with this conclusion is inhibition of the chemotactic response to monocytes by agents that block CXCR2, the receptor that NAP-2 uses. Later, when the monocyte moves from the center of chemoattraction, the activated PMN themselves, whose own chemotactic properties are enhanced in EDTA/plasma, appear to take over generation of the gradient, resulting in a prolonged ingress of PMN from outside the field ("second wave"). Chemoattraction by monocytes seems to be simply one way of stimulating the PMN, which, once activated, fail in EDTA/plasma to efficiently shut off their own chemoattraction for other PMN. We suggest that these exaggerated chemotactic effects are due to the loss of normal modulation by a regulatory factor(s) designed to keep the chemotactic response from getting out of hand-i.e., a tonic inhibitor of chemotaxis in plasma.

Recruitment of the cross-linked opsonic receptor CD32A (FcgammaRIIA) to high-density detergent-resistant membrane domains in human neutrophils.

We have previously shown that CD32A (or FcgammaRIIA), one of the main opsonin receptors, was rapidly insolubilized and degraded in intact neutrophils after its cross-linking. In view of these experimental difficulties, the early signalling steps in response to CD32A activation were studied in purified plasma membranes of neutrophils. After CD32A cross-linking in these fractions, the tyrosine phosphorylation of two major substrates, the receptor itself and the tyrosine kinase Syk, was observed. Phosphorylation of these two proteins was observed only in the presence of orthovanadate, indicating the presence, in the membranes, of one or more tyrosine phosphatases that maintain CD32A dephosphorylation. The tyrosine phosphorylation of these two proteins was inhibited by the Src kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). The ligation of CD32A led to its recruitment to a previously uncharacterized subset of high-density flotillin-1-positive DRMs (detergent-resistant membranes). The changes in the solubility properties of CD32A were observed in the absence of added ATP; therefore, they were probably not secondary to the tyrosine phosphorylation of the receptor, rather they preceded it. Src kinases as well as Syk were constitutively present in DRMs of high and low density and no evident changes in their distribution were detected after cross-linking of CD32A. Pretreatment of plasma membranes with methyl-beta-cyclodextrin did not inhibit the recruitment of CD32A to DRMs, although it led to the loss of the Src kinase Lyn from these fractions. In addition, methyl-beta-cyclodextrin inhibited the tyrosine phosphorylation of CD32A and Syk induced by cross-linking of CD32A. This membrane model allowed us to observe a movement of CD32A from detergent-soluble regions of the membranes to DRMs, where it joined Src kinases and Syk and became tyrosine-phosphorylated.

Misoprostol Stimulates cAMP Generation in Human Leukocytes: Synergy with Colchicine Suggests a New Potential for Established Drugs.

In isolated leukocytes, elevation of cAMP can inhibit various proinflammatory and immune functions. The prostaglandins E (PGEs) are known to stimulate leukocyte cAMP production, and for years they have been viewed as potential immunosuppressive and/or anti-inflammatory agents. However, their clinical use is severely limited by extreme metabolic instability and by poor oral absorption, which necessitates administration by infusion or injection. Misoprostol is a synthetic analog of PGE(1) that is relatively stable and orally absorbable. We examined the effects of misoprostol on cAMP production in leukocytes, in view of the possibility that it mimics PGE(1) and, thus, might represent a clinically useful immunosuppresive or anti-inflammatory drug. Our results indicate the following: (1) Misoprostol increases leukocyte cAMP production in a dose-dependent manner (similar20 nM to >100 &mgr;M) and acts by stimulating adenylate cyclase. (2) Its potency and maximal effect are somewhat less than those of PGE(1) (3) cAMP generation in response to either misoprostol or PGE(1) is transient (in the presence of isobutylmethylxanthine to inhibit endogenous phosphodiesterases). (4) Misoprostol's stimulation of adenylate cyclase is synergistically increased by pretreatment of cells with colchicine, a microtubule-disrupting agent that is currently used for prophylaxis and treatment of gout. (5) Colchicine acts by increasing the initial rate of cAMP production and not by prolonging the response to misoprostol. (6) A clinically relevant dose of colchicine (0.25 &mgr;M) is effective given sufficient pretreatment time. (7) Whereas a clinically relevant dose of misoprostol (3 nM) is ineffective alone, preexposure of cells to colchicine enables such a dose to stimulate cAMP generation significantly. The combination of misoprostol with colchicine might eventually prove useful in the therapy of immune or inflammatory disease.

Reduced bioenergetics and toll-like receptor 1 function in human polymorphonuclear leukocytes in aging.

Aging is associated with a progressive decline in immune function (immunosenescence) resulting in an increased susceptibility to viral and bacterial infections. Here we show reduced expression of Toll-like receptor 1 (TLR1) in polymorphonuclear leukocytes (PMN) and an underlying age-dependent deficiency in PMN bioenergetics. In older (>65 years) adults, stimulation through TLR1 led to lower activation of integrins (CD11b and CD18), lower production of the chemokine IL-8, and lower levels of the phosphorylated signaling intermediate p38 MAP kinase than in PMN from younger donors (21-30 years). In addition, loss of CD62L, a marker of PMN activation, was reduced in PMN of older adults stimulated through multiple pathways. Rescue of PMN from apoptosis by stimulation with TLR1 was reduced in PMN from older adults. In seeking an explanation for effects of aging across multiple pathways, we examined PMN energy utilization and found that glucose uptake after stimulation through TLR1 was dramatically lower in PMN of older adults. Our results demonstrate a reduction in TLR1 expression and TLR1-mediated responses in PMN with aging, and reduced efficiency of bioenergetics in PMN. These changes likely contribute to reduced PMN efficiency in aging through multiple aspects of PMN function and suggest potential therapeutic opportunities.

Plunder of Human Blood Leukocytes Containing Ingested Material, by Other Leukocytes: Where Is the Fusagen That Allows Preservation of Membrane Integrity and Motile Function?

In studying phagocytosis of zymosan particles by human blood monocytes in phase-contrast videomicroscopy, we found that monocytes loaded with zymosan particles became chemotactic for polymorphonuclear leukocytes (PMN) which closed on them and purloined their particle content. This despoliation usually occurred in monocytes that had begun to swell-prefiguring their death. The violent seizure of their contents by the aggressing PMN often tore the monocytes apart. However, some apparently healthy monocyte survived the removal of zymosan content by PMN or, more commonly, its removal by another monocyte. PMN-a much hardier cell in slide preparations-that were similarly loaded with zymosan particles, also attracted PMN. The latter could remove zymosan from the target cell without killing it. Thus, leukocytes were sacrificing significant portions of themselves without losing residual membrane integrity and motile function. Their behavior with respect to other particles (e.g., bacteria) will be of interest. We suggest that the membrane fusagen resides in the inner membrane leaflets when they are brought together in an extreme hourglass configuration. This event may be similar to the fragmentation of erythrocytes into intact pieces, the formation of cytokineplasts, the rear extrusion of content by migrating cells on surfaces, and the phagocytic process itself.

Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo.

Neutrophil extracellular traps (NETs) are released as neutrophils die in vitro in a process requiring hours, leaving a temporal gap that invasive microbes may exploit. Neutrophils capable of migration and phagocytosis while undergoing NETosis have not been documented. During Gram-positive skin infections, we directly visualized live polymorphonuclear cells (PMNs) in vivo rapidly releasing NETs, which prevented systemic bacterial dissemination. NETosis occurred during crawling, thereby casting large areas of NETs. NET-releasing PMNs developed diffuse decondensed nuclei, ultimately becoming devoid of DNA. Cells with abnormal nuclei showed unusual crawling behavior highlighted by erratic pseudopods and hyperpolarization consistent with the nucleus being a fulcrum for crawling. A requirement for both Toll-like receptor 2 and complement-mediated opsonization tightly regulated NET release. Additionally, live human PMNs injected into mouse skin developed decondensed nuclei and formed NETS in vivo, and intact anuclear neutrophils were abundant in Gram-positive human abscesses. Therefore early in infection NETosis involves neutrophils that do not undergo lysis and retain the ability to multitask.

Anaplasma phagocytophilum induces actin phosphorylation to selectively regulate gene transcription in Ixodes scapularis ticks.

Anaplasma phagocytophilum, the agent of human anaplasmosis, persists in ticks and mammals. We show that A. phagocytophilum induces the phosphorylation of actin in an Ixodes ricinus tick cell line and Ixodes scapularis ticks, to alter the ratio of monomeric/filamentous (G/F) actin. A. phagocytophilum-induced actin phosphorylation was dependent on Ixodes p21-activated kinase (IPAK1)-mediated signaling. A. phagocytophilum stimulated IPAK1 activity via the G protein-coupled receptor Gbetagamma subunits, which mediated phosphoinositide 3-kinase (PI3K) activation. Disruption of Ixodes gbetagamma, pi3k, and pak1 reduced actin phosphorylation and bacterial acquisition by ticks. A. phagocytophilum-induced actin phosphorylation resulted in increased nuclear G actin and phosphorylated actin. The latter, in association with RNA polymerase II (RNAPII), enhanced binding of TATA box-binding protein to RNAPII and selectively promoted expression of salp16, a gene crucial for A. phagocytophilum survival. These data define a mechanism that A. phagocytophilum uses to selectively alter arthropod gene expression for its benefit and suggest new strategies to interfere with the life cycle of this intracellular pathogen, and perhaps other Rickettsia-related microbes of medical importance.

Increased TLR4 expression and downstream cytokine production in immunosuppressed adults compared to non-immunosuppressed adults.

BACKGROUND: An increasing number of patients have medical conditions with altered host immunity or that require immunosuppressive medications. While immunosuppression is associated with increased risk of infection, the precise effect of immunosuppression on innate immunity is not well understood. We studied monocyte Toll-like receptor (TLR) expression and cytokine production in 137 patients with autoimmune diseases who were maintained on immunosuppressive medications and 419 non-immunosuppressed individuals. METHODOLOGY/PRINCIPAL FINDINGS: Human peripheral blood monocytes were assessed for surface expression of TLRs 1, 2, and 4. After incubation with TLR agonists, in vitro production of the cytokines IL-8, TNFalpha, and MIF were measured by ELISA as a measure of TLR signaling efficiency and downstream effector responsiveness. Immunosuppressed patients had significantly higher TLR4 surface expression when compared to non-immunosuppressed adults (TLR4 %-positive 70.12+/-2.28 vs. 61.72+/-2.05, p = 0.0008). IL-8 and TNF-alpha baseline levels did not differ, but were significantly higher in the autoimmune disease group following TLR stimulation. By contrast, baseline MIF levels were elevated in monocytes from immunosuppressed individuals. By multivariable analyses, IL-8 and TNFalpha, but not MIF levels, were associated with the diagnosis of an underlying autoimmune disease. However, only MIF levels were significantly associated with the use of immunosuppressive medications. CONCLUSIONS/SIGNIFICANCE: Our results reveal that an enhanced innate immune response is a feature of patients with autoimmune diseases treated with immunosuppressive agents. The increased risk for infection evident in this patient group may reflect a dysregulation rather than a simple suppression of innate immunity.

Clocking the Lyme spirochete.

In order to clear the body of infecting spirochetes, phagocytic cells must be able to get hold of them. In real-time phase-contrast videomicroscopy we were able to measure the speed of Borrelia burgdorferi (Bb), the Lyme spirochete, moving back and forth across a platelet to which it was tethered. Its mean crossing speed was 1,636 microm/min (N = 28), maximum, 2800 microm/min (N = 3). This is the fastest speed recorded for a spirochete, and upward of two orders of magnitude above the speed of a human neutrophil, the fastest cell in the body. This alacrity and its interpretation, in an organism with bidirectional motor capacity, may well contribute to difficulties in spirochete clearance by the host.

Recruitment of macrophages and polymorphonuclear leukocytes in Lyme carditis.

Lyme arthritis, caused by the spirochete Borrelia burgdorferi, can be recurrent or prolonged, whereas Lyme carditis is mostly nonrecurring. A prominent difference between arthritis and carditis is the differential representation of phagocytes in these lesions: polymorphonuclear leukocytes (PMN) are more prevalent in the joint, and macrophages predominate in the heart lesion. We have previously shown differential efficiency of B. burgdorferi clearance by PMN and macrophages, and we now investigate whether these functional differences at the cellular level may contribute to the observed differences in organ-specific pathogenesis. When we infected mice lacking the neutrophil chemokine receptor (CXCR2(-/-) mice) with spirochetes, we detected fewer PMN in joints and less-severe arthritis. Here we have investigated the effects of the absence of the macrophage chemokine receptor CCR2 on the development and resolution of Lyme carditis in resistant (C57BL/6J [B6]) and sensitive (C3H/HeJ [C3H]) strains of mice. In B6 CCR2(-/-) mice, although inflammation in hearts is mild, we detected an increased burden of B. burgdorferi compared to that in wild-type (WT) mice, suggesting reduced clearance in the absence of macrophages. In contrast, C3H CCR2(-/-) mice have severe inflammation but a decreased B. burgdorferi burden compared to that in WT C3H mice both at peak disease and during resolution. Histopathologic examination of infected hearts revealed that infected C3H CCR2(-/-) animals have an increased presence of PMN, suggesting compensatory mechanisms of B. burgdorferi clearance in the hearts of infected C3H CCR2(-/-) mice. The more efficient clearance of B. burgdorferi from hearts by CCR2(-/-) versus WT C3H mice suggests a natural defect in the recruitment or function of macrophages in C3H mice, which may contribute to the sensitivity of this strain to B. burgdorferi infection.

Human neutrophil calprotectin reduces the susceptibility of Borrelia burgdorferi to penicillin.

Borrelia burgdorferi, the spirochetal agent of Lyme disease, is susceptible to killing by a variety of polymorphonuclear leukocyte (PMN) components. Some are most effective against metabolically active B. burgdorferi. The abundant PMN cytoplasmic protein calprotectin, elevated 10- to 100-fold in inflammation, inhibits the growth of spirochetes through chelation of the essential cation, Zn. Since the action of some therapeutic antibiotics depends on bacterial division, we investigated the antibiotic sensitivities of spirochetes in calprotectin. In physiologic calprotectin, B. burgdorferi is not eliminated by therapeutic doses of penicillin G; in contrast, doxycycline is effective. Calprotectin may modify the clearance of spirochetes at sites of inflammation.

Cryopreservable neutrophil surrogates: granule-poor, motile cytoplasts from polymorphonuclear leukocytes home to inflammatory lesions in vivo.

Cytokineplasts (CKP) are anucleate, motile, granule-poor fragments induced from polymorphonuclear leukocytes on surfaces by the brief application of heat. Derived from the peripheral cytoplasm and membranes of PMN, they retain the sensing, transducing, and effector mechanisms necessary for chemotaxis and phagocytosis, and appear to represent a functional, self-purification of the motile apparatus. Unlike their parent PMN, CKP are cryopreservable. We have shown that they can adhere to endothelial cell monolayers, open interendothelial cell junctions, and migrate to the abluminal side when stimulated by a chemoattractant. Employing an animal model, we now show that, given intravenously, they can home to an inflammatory target lesion in vivo.

Calprotectin, an abundant cytosolic protein from human polymorphonuclear leukocytes, inhibits the growth of Borrelia burgdorferi.

We previously showed that numerous polymorphonuclear leukocyte (PMN) granule components efficiently kill Borrelia burgdorferi, the agent of Lyme disease. In addition, motile, granule-poor cytoplasts (U-Cyt) from human blood PMN can exert anti-Borrelia activity against opsonized B. burgdorferi independently of oxidative mechanisms. Here we show that lysates of U-Cyt also possess anti-Borrelia activity, a portion of which comes from the abundant cytosolic protein calprotectin. The anti-Borrelia activity of U-Cyt lysates and recombinant calprotectin was partially or completely reversed by specific antibody to calprotectin and by Zn(2+), a cation essential for the growth of B. burgdorferi and known to inhibit the antimicrobial activity of calprotectin. Quantitative microscopic and regrowth assays revealed that calprotectin acted in a bacteriostatic fashion against B. burgdorferi. We conclude that calprotectin, a potent bacteriostatic agent from a cell primarily recognized for its oxidative and granular antibacterial mechanisms, may play a modulatory role in infection by the Lyme spirochete, particularly at sites of acute inflammation.

Borrelia burgdorferi are susceptible to killing by a variety of human polymorphonuclear leukocyte components.

The killing of Borrelia burgdorferi by intact human polymorphonuclear leukocytes (PMNL) and by individual PMNL components was compared. Intact PMNL killed B. burgdorferi 6.5-fold more efficiently and 5-fold more completely when spirochetes were opsonized with specific antibodies. U-cytoplasts, which have activatable oxidase, killed opsonized B. burgdorferi with an efficiency similar to that of intact PMNL in killing unopsonized B. burgdorferi. Although B. burgdorferi were susceptible to H(2)O(2) and nitric oxide, PMNL lysates killed B. burgdorferi nearly as well as intact PMNL killed opsonized B. burgdorferi, suggesting a critical role for granule contents. B. burgdorferi were killed by the PMNL antimicrobial components elastase, LL-37, bactericidal/permeability-increasing protein, and human neutrophil peptide-1. B. burgdorferi had limited susceptibility to killing by lysozyme and were not killed by azurocidin, proteinase 3, or lactoferrin. The efficient killing of B. burgdorferi by a variety of PMNL mechanisms highlights the paradoxical persistence of spirochetes in vivo.

Tick saliva reduces adherence and area of human neutrophils.

During natural infection with the agent of Lyme disease, Borrelia burgdorferi, spirochetes are delivered with vector saliva, which contains anti-inflammatory and antihemostatic activities. We show here that the saliva of ixodid ticks reduces polymorphonuclear leukocyte (PMN) adhesion via downregulation of beta2-integrins and decreases the efficiency of PMN in the uptake and killing of spirochetes. Inhibition of integrin adhesion and signaling reduces anti-inflammatory functions of PMN. These effects may favor the initial survival of spirochetes in vivo.

Human phagocytic cells in the early innate immune response to Borrelia burgdorferi.

During natural infection with the agent of Lyme disease, Borrelia burgdorferi, polymorphonuclear leukocytes (PMNL) are the first cells of the innate immune system to arrive at the site of spirochete deposition in the skin. This study examined the degree of spirochete clearance likely to occur with PMNL or mononuclear cells before the development of the secondary immune response. Without specific antibody in vitro, there was very limited uptake of spirochetes by PMNL or monocytes and no intracellular colocalization of PMNL granule products with spirochetes. Most of the killing of spirochetes by PMNL was extracellular. In contrast, mature macrophages ingest and kill spirochetes avidly with or without specific antibody. Once the spirochetes are opsonized, PMNL clear them rapidly. These findings may be relevant to the initial survival of spirochetes introduced into the host.